Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalyticall
Trang 1glucokinase – nucleotide-induced conformational changes with possible implications for its kinetic cooperativity
Janne Molnes1,2,3, Knut Teigen3, Ingvild Aukrust1,2,3, Lise Bjørkhaug2,4, Oddmund Søvik2, Torgeir Flatmark3and Pa˚l Rasmus Njølstad1,2
1 Department of Pediatrics, Haukeland University Hospital, Bergen, Norway
2 Department of Clinical Medicine, University of Bergen, Norway
3 Department of Biomedicine, University of Bergen, Norway
4 Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Norway
Introduction
Glucokinase (GK) or hexokinase IV (EC 2.7.1.1)
catal-yses the phosphorylation of a-d-glucose (Glc) to form
glucose 6-phosphate, the entry point of Glc into
gly-colysis, using MgATP2) as the phosphoryl donor
Human GK (hGK) is expressed in the liver [1], pan-creas [2], brain, and endocrine cells of the gut [3,4] It
is a key regulatory enzyme in the human pancreatic b-cell (isoform 1), playing a crucial role in the regulation
Keywords
ATP binding; catalytic mechanism; GCK
maturity onset diabetes of the young
(GCK-MODY); glucokinase; kinetic cooperativity
Correspondence
T Flatmark, Department of Biomedicine,
University of Bergen, N-5009 Bergen,
Norway
Fax: +47 55586360
Tel: +47 55586428
E-mail: torgeir.flatmark@biomed.uib.no
Note
The atomic coordinates of the molecular
dynamics simulated structural models are
available from knut.teigen@biomed.uib.no
(Received 7 April 2011, revised 20 April
2011, accepted 4 May 2011)
doi:10.1111/j.1742-4658.2011.08160.x
Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic b-cells, and catalytic activation by a-D-glucose binding has
a key regulatory function Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand-free enzyme and how
it affects its conformation Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect Adeno-sine-5¢-(b,c-imido)triphosphate and ATP bind to the wild-type enzyme with apparent [L]0.5 (ligand concentration at half-maximal effect) values of 0.27 ± 0.02 mM and 0.78 ± 0.14 mM, respectively The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate and limited proteol-ysis by trypsin, and by molecular dynamic simulations The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface⁄ active site loop and partial closure
of the active site cleft In the complex, the adenosine moiety is packed between two a-helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415) Combined with enzyme kinetic analyses, our data indicate that the ATP-induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme
Abbreviations
AdN, adenine nucleotide; AMP-PNP, adenosine-5¢-(b,c-imido)triphosphate; ANS, 8-anilinonaphthalene-1-sulfonate; ATPcS, adenosine-5¢-O-(3-thiotriphosphate); GCK-MODY, GCK maturity-onset diabetes of the young; GK, glucokinase; GKA, glucokinase activator; Glc, a- D -glucose; GST, glutathione-S-transferase; hGK, human glucokinase; ITF, intrinsic tryptophan fluorescence; MD, molecular dynamic; n H , Hill coefficient; PDB, Protein Data Bank; WT, wild-type.
Trang 2of insulin secretion, and is therefore termed the
pancre-atic b-cell glucose sensor [5] In humans, more than
600 different mutations in the glucokinase gene (GCK)
have been detected in patients suffering from familial,
mild fasting hyperglycaemia [GCK maturity onset
dia-betes of the young (GCK-MODY), GCK permanent
neonatal diabetes mellitus, and GCK congenital
hyper-insulinism of infancy [6–11] Some of the mutations
greatly reduce the binding affinity of MgATP2)
[11,12], which is compatible with a direct interaction of
these residues with the nucleotide at the active site
The catalytic mechanism of GK has been the subject
of several detailed analyses, and is still a partly
unre-solved issue Although some theoretical evidence has
been presented in support of a random order
mecha-nism, in which the enzyme interacts with the substrate
and cosubstrate in a random fashion [13], enzyme
kinetic studies support an ordered mechanism in which
Glc binds to the enzyme before the cosubstrate [14–
16] The discussion is reminiscent of that related to the
catalytic mechanism of yeast hexokinase [17] For both
enzymes, part of the discussion has been related to the
question of whether ATP binds to the Glc-free enzyme
and the possibility of a nucleotide-triggered change in
protein conformation
In this work, we have studied the interaction of
ATP and analogues with the human pancreatic enzyme
with the aims of: (a) presenting experimental evidence
for equilibrium binding to the ligand-free super-open
conformation; (b) demonstrating possible
conforma-tional changes associated with ATP binding; (c)
obtaining insights into the active site contact residues
involved in ATP binding; and (d) relating this
informa-tion to steady-state enzyme kinetic data To achieve
these aims, we used a combined experimental approach
including intrinsic tryptophan fluorescence (ITF),
extrinsic 8-anilino-1-naphthalenesulfonate (ANS)
fluo-rescence, limited proteolysis, and molecular dynamic
(MD) simulations Additionally, enzyme kinetic
analy-ses were performed to evaluate the functional
implica-tions of the structural data The different approaches
provide new insights into the interaction of ATP with
hGK, with possible implications for the positive kinetic
cooperativity with respect to Glc
Results
Recombinant proteins
The average yields of soluble recombinant pancreatic
glutathione-S-transferase (GST)–hGK fusion proteins
were 4.0 mg L)1 (wild type and T228M) and
2.0 mg L)1 (L146R) As the recombinant wild-type
(WT) hGK and WT GST–hGK enzymes demonstrate similar steady-state kinetic parameters and the same apparent Kd for Glc in the ITF equilibrium binding assay [18], the fusion proteins were used in kinetic studies and ITF equilibrium binding analyses with Glc
In the adenine nucleotide (AdN) equilibrium binding studies, we compared nontagged and GST-tagged GK
In all other experiments, only the nontagged proteins were used
Characterization of the T228M mutant reference enzyme
The T228M mutant form, causing GCK-MODY in the heterozygous state and GCK permanent neonatal dia-betes mellitus in the homozygous state [9,19], was selected as a non-ATP-binding reference enzyme on the basis of its previously described kinetic properties [9,20,21] Here, equilibrium binding of Glc, as deter-mined by ITF, demonstrated an increased affinity (Kd= 3.1 ± 0.1 mm) in comparison with WT GST– hGK (Kd= 4.3 ± 0.1 mm), and a fluorescence enhancement signal response [(DFeq⁄ F0)max· 100] simi-lar to that of the wild type (Table 1) Steady-state kinetic analyses demonstrated a 9000-fold reduced catalytic activity (kcat 7 · 10)3s)1) (Table 1) Thr228 is a highly conserved residue at the active site
of the hexokinase family of enzymes, positioned in the phosphate-binding loop and part of a classical ATP-binding motif (phosphate 2 site) in hexokinases and homologous proteins [22] In the crystal structures of
Table 1 The steady-state kinetics and ITF properties of WT GST– hGK and two GCK-MODY mutant forms NM, not measurable.
kcat(s)1) c 67.6 ± 1.3 7 · 10)3 0.77 ± 0.03
Relative catalytic activity (%)
KmMgATP2)(m M ) 0.16 ± 0.01 NM 0.24 ± 0.04 Hill coefficient (nH) c 1.95 ± 0.19 NM 1.29 ± 0.04 Hill coefficient (n H ) d 1.15 ± 0.04 NM 0.73 ± 0.04 Glc response (%)
[(DFeq⁄ F o )max· 100]
28.7 ± 1.5 29.2 ± 0.1 5.3 ± 0.5
K d Glc (m M ) e 4.3 ± 0.1 3.1 ± 0.1 19.3 ± 3.8
a The n H , [S] 0.5 and K d values were not measured, because of low catalytic activity. bThe ITF responses to 200 m M Glc were 33.2 and 36.0 arbitrary fluorescence units for the fusion protein and the isolated T228M hGK mutant, respectively c Assay with Glc as the variable substrate. dAssay with ATP as the variable substrate.
e Obtained from equilibrium binding measurements by intrinsic Trp fluorescence spectroscopy.
Trang 3human and yeast hexokinases, the hydroxyl group of
this conserved Thr interacts with the a-phosphate of
ATP [21,23,24], and a Thrfi Met substitution in hGK
is inferred to eliminate this important contact (see the
in silico studies below) According to the coordinates
of the closed (Glc-bound) conformation of WT hGK
[Protein Data Bank (PDB) ID 1v4s], the T228M
mutation is predicted to be destabilizing, as measured
by the free energy of thermal unfolding (DDG =
)4.07 kcalÆmol)1) and the free energy of folding
(DDG = 0.85 kcalÆmol)1) However, the far-UV CD
spectrum was very similar, if not identical, to that of
WT hGK (Fig S1), and no significant differences in
the apparent Tmvalues (on thermal unfolding) of WT
hGK (Fig 1) and the mutant protein (data not shown)
were observed Thus, the Thrfi Met substitution has
little impact on the protein fold
Equilibrium binding of adenosine-5¢-(b,c-imido)
triphosphate (AMP-PNP), ATP and MgATP to the
ligand-free enzyme
To study binding of AdNs to the ligand-free nontagged
enzyme, we first measured the change in ITF
[(DFeq⁄ F0)· 100] at 25 C as a function of the AdN
concentration In contrast to the enhancement of the
ITF signal observed with Glc [18,25], the ATP analogue
AMP-PNP resulted in quenching of the fluorescence
(Fig 2), consistent with a previous report [26] However,
the inner filter effect resulting from nucleotide
absor-bance at the excitation wavelength (295 nm), which was
not considered in that report, made a significant
contri-bution to the quenching To correct for this effect, a
sim-ilar titration was performed with the non-ATP-binding
mutant T228M and with free Trp (Fig 2A,C) Of the
two reference titrations, the T228M mutant gave the
preferred correction (Fig 2A), as the mutant also
dem-onstrated quenching of the ITF at low concentrations
(£ 0.1 mm) From the fluorescence difference data
(Fig 2B), an apparent [L]0.5 (ligand concentration at
half-maximal effect) value of 0.27 ± 0.02 mm (25C)
was estimated by nonlinear regression analysis The net
(specific) fluorescence quenching observed for AMP-PNP
was modest, but significant [D(DFeq⁄ F0)max· 100 =
)2.6% ± 0.2%], suggesting that one or more of the
three Trp residues (Trp99, Trp167, and Trp257) undergo
small changes in quantum yield, but without any
signifi-cant spectral shift A similar result was obtained with
the respective GST–hGK fusion proteins (Fig 2C,D),
with an [L]0.5value of 0.16 ± 0.04 mm and D(DFeq⁄ F0
)-max· 100 =)2.2% ± 0.2% In the ITF titrations of
the wild type and the T228M mutant (control) with
increasing concentrations of ATP (Fig 2E), a net
decrease in fluorescence intensity similar to the AMP-PNP response was observed The differential binding data (Fig 2E) were fitted to a hyperbolic binding iso-therm by nonlinear regression (r2> 0.97), giving a half-maximal effect ([L]0.5) at 0.78± 0.14 mm and D(DFeq⁄ F0)max · 100 =)1.5% ± 0.1% Similar titrations with MgATP gave comparable maximal quenching of ITF of D(DFeq⁄ F0)max· 100= )2.2% ± 0.3%
A
B
Fig 1 Thermal refolding–unfolding and aggregation of WT hGK The experiments were performed as described in Experimental pro-cedures (A) The thermal refolding–unfolding profile of WT hGK (23 l M ) in the absence of Glc was determined by following the change in ellipticity at 222 nm at a constant heating rate of
40 CÆh)1 An apparent transition temperature (T m ) of 42.4 ± 0.2 C was determined from the first derivative of the smoothed denatur-ation curve No significant difference in the profile was observed in the presence of Glc (data not shown) The observed optical activity
is expressed as the mean residue molar ellipticity ([h]MR) (B) The pseudo-absorbance data were obtained at the same time as the
CD data in (A), reporting on the biphasic heat-induced increase in absorbance The regression lines, based on data points in the tem-perature interval 24–79 C, indicate an inflection point at 42 C and increasing aggregation of the protein above this temperature; above 80 C, the absorbance decreased, probably owing to pre-cipitation of the protein.
Trang 4Thermal refolding and unfolding
As previously demonstrated by ITF, ligand-free WT
hGK senses temperature shifts from 4 to 39C directly
by a slow (seconds to minutes) conformational change
(hysteresis), with a biphasic time course in temperature
jump (4–39C) experiments [18] The far-UV CD
spec-troscopy at 222 nm confirmed this conformational
change by an apparent change in the secondary
struc-ture in the same temperastruc-ture range (Fig 1A) At
higher temperatures, the enzyme demonstrated
rela-tively low global thermodynamic stability, with an
apparent Tmof 42.4 ± 0.2C and increasing
aggrega-tion at temperatures ‡ 42 C, as measured from the
associated high-voltage (pseudo-absorbance) curve
obtained at the same time (Fig 1B) Similarly, the iso-thermal (25C) chemical unfolding caused by guani-dine chloride also resulted in aggregation of the protein (data not shown) This instability of the pro-tein precluded an estimate of equilibrium thermody-namic parameters, and thus also measurement of the effect of ligands on such conformational equilibria
Effect of ATP and Glc on extrinsic ANS fluorescence and limited proteolysis ANS is an extrinsic fluorophore with affinity for hydro-phobic clusters in proteins that are not tightly packed
in a fully folded structure or become exposed in par-tially unfolded structures [27] The weak fluorescence of
[AMP-PNP] (m M )
WT hGK T228M hGK Tryptophan
A
0.0 0.5 1.0 1.5 2.0 2.5 3.0
) ] x 100
[AMP-PNP] (m M )
B
1.0 2.0 3.0 4.0 5.0
[AMP-PNP] (m M )
WT GST-hGK T228M GST-hGK Tryptophan
GST–hGK
C
[AMP-PNP] (m M )
) ] x 100
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
D
) ] x 100
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
[ATP] (m M )
E
Fig 2 Equilibrium binding of AMP-PNP (A–D) and ATP (E) in the absence of Glc (A) The change in fluorescence intensity [(DFeq⁄ F 0 ) · 100] was measured at 25 C upon subsequent additions of ligand (A) AMP-PNP titration curves of WT hGK (d), the non-ATP-binding mutant T228M hGK (s), and free Trp (at a concentration giving the same F 0 value as the enzyme) (.) The data were fitted to binding isotherms by nonlinear regression analysis, with r 2 > 0.99 for both WT hGK and T228M hGK Data points and error bars represent the mean ± SD of three independent titrations (B) The net fluorescence quenching [D(DFeq⁄ F 0 )max· 100] of WT hGK as a function of [AMP-PNP], with a calculated [L] 0.5 value of 0.27 ± 0.02 m M The data points and the solid line represent the difference between the WT and T228M hGK titrations (C) The same experiment as in (A), but performed on the GST fusion proteins The titration curves of WT GST-hGK (d), the non-ATP-binding mutant T228M GST–hGK (s), and free Trp (at a concentration giving the same F0value as the enzyme) (.) The data were fitted to binding isotherms by nonlinear regression analysis, with r 2 > 0.99 for both WT GST–hGK and T228M GST–hGK (D) The net fluorescence quenching [D(DF eq ⁄ F 0 ) max · 100] of WT GST–hGK as a function of [AMP-PNP], with a calculated [L] 0.5 value of 0.16 ± 0.04 m M The data points and the solid line represent the difference between the WT and T228M GST–hGK titrations (E) Equilibrium binding of ATP to WT GST–hGK in the absence of Glc The figure shows the net decrease in ITF [D(DF eq ⁄ F 0 ) max · 100] with increasing concentrations of ATP (25 C), calculated
in a similar manner as in (B) and (D), representing the difference between the WT GST–hGK and T228M GST–hGK titrations The data were fitted to a hyperbolic binding isotherm by nonlinear regression analysis (r 2 > 0.97), and an [L]0.5value for ATP of 0.78 ± 0.14 m M was calcu-lated The data points (d) represent the means of duplicate titration experiments.
Trang 5ANS was greatly enhanced upon binding to ligand-free
WT hGK (Fig 3A), with a maximum at 480 nm
(blue shift), indicative of ANS binding to exposed
hydrophobic clusters As seen from Fig 3B, both ATP
and Glc significantly reduced the ANS fluorescence
sig-nal [Glc (P = 0.00004) > ATP (P = 0.004)],
compati-ble with a decrease in accessicompati-ble hydrophobic clusters
as compared with the ligand-free enzyme
In our studies on mutant forms of hGK, their
sus-ceptibilities to limited proteolysis by trypsin have
proved to be a valuable conformational probe
(unpub-lished data) Here, it was demonstrated (Fig 3C) that
the ligand-free WT hGK (at 25C) is partly stabilized
by its association with ATP and Glc (Glc > ATP)
Effect of nonhydrolysable ATP analogues on the
equilibrium binding of Glc
The equilibrium binding of Glc to the ligand-free WT
hGK and its binary AdN complexes was determined
by its enhancement of the ITF signal (Table 2) In the
absence of AdNs, a hyperbolic binding isotherm for
Glc was observed, with a Kdvalue of 4.2 ± 0.1 mm at
25C Titration with Glc in the presence of
Mg-adeno-sine-5¢-O-(3-thiotriphosphate) (ATPcS) and
MgAMP-PNP also gave hyperbolic binding isotherms; however,
the apparent affinity for Glc increased (Table 2), i.e
about two-fold with 5 mm MgAMP-PNP (P = 0.002)
A similar effect was observed for the GCK-MODY
L146R mutant in the presence of 2.5 mm ATPcS; that
is, the apparent Kd decreased from 19.3 ± 3.8 mm to
14.0 ± 1.4 mm (Fig 4), and there was a 25%
incre-ase in the fluorescence signal response [(DFeq⁄ F0)max·
100] The mutant demonstrated a 100-fold reduction
in kcatand a 40-fold increase in the [S]0.5(substrate
concentration at half-maximal activity) value for Glc
(Table 1) The positive kinetic cooperativity with
respect to Glc was partly lost in the mutant
(nH= 1.29 ± 0.04), and in contrast to previous
find-ings [28], the Km for ATP (0.24 ± 0.04 mm) was only
slightly increased
In silico dynamic and conformational effects of
ATP binding
In the MD simulations, the starting crystal structure
(PDB ID 1v4t) of the ligand-free super-open
confor-mation was modified to include the 23 missing residues
(Glu157–Asn179) in a surface loop structure (see
Experimental procedures) The Ca rmsd value for the
modelled structure and the crystal structure was
2.3 A˚ when the Glu157–Asn179 loop residues were
not included From the computed B-factor values (Figs
A
B
C
Fig 3 ANS fluorescence measurements and limited proteolysis (A) Emission fluorescence spectra (k ex = 385 nm) of free ANS in buffer and ANS in the presence of 0.75 l M WT hGK A final ANS concentration of 60 l M was used (B) The effect of ATP and Glc on ANS binding to WT hGK The ANS binding experiments were per-formed at a temperature of 38 C, as described in Experimental procedures, with 60 l M ANS and a protein concentration of 0.75 l M The concentrations of Glc and ATP were 30 m M and
2 m M , respectively Each column represents the mean ± SD of three independent experiments Statistical significance was deter-mined with Student’s t-test: **P < 0.01 and ***P < 0.0001 (C) Time-course for the limited proteolysis of WT hGK by trypsin WT GST–hGK (0.5 mgÆmL)1) was cleaved with factor Xa for 2 h at 4 C, and subsequently subjected to limited proteolysis by trypsin at
25 C (trypsin ⁄ hGK ratio of 1 : 400 by mass) in the absence of ligand (d), or in the presence of either 40 m M Glc ( ) or 2 m M ATP ⁄ 4 m M MgAc (s) Data points and error bars represent the mean ± SD of three independent experiments.
Trang 65A and S2B), the region that fluctuates the most is
Glu157–Asn179, consistent with the observed disorder
in the crystal structure MD simulations of the
mod-elled binary GK–ATP complex revealed that the global
rmsd of the structure converged at the end of the 2-ns
simulation period (Fig S2A) The dynamic changes in
the active site cleft opening over the 2-ns equilibration
period (Fig 5C), as defined by the residues Lys169–
Gly223 (‘hinge’)–Gly229, suggest partial closure of the
interdomain cleft ( 15) These defining residues were
previously used to monitor the opening of the cleft
Table 2 The effect of ATP analogues on the equilibrium binding
affinity of Glc as determined by ITF fluorescence titrations on WT
GST–hGK.
MgAMP-PNP
MgATPcS
a Mean ± SD of five independent titration experiments b Based on
nonlinear regression analysis of single binding isotherms (r2> 0.99)
(n = 12 data points) c Mean ± SD of three independent titration
experiments.
Fig 4 The Glc binding isotherm for the mutant L146R GST–hGK.
The enhancement of ITF was measured at 25 C with increasing
concentrations of Glc in the absence (d) and presence (s) of
2.5 m M ATPcS The solid lines represent the fit of the data to two
hyperbolas as obtained by nonlinear regression analyses, giving Kd
values of 19.3 ± 3.8 m M (r 2 > 0.98) and 14.0 ± 1.4 m M (r 2 > 0.99)
in the absence and presence of ATPcS, respectively, and a
fluores-cence signal response [(DF eq ⁄ F 0 ) max · 100] of 5% For
compari-son, the (DFeq⁄ F 0 )max· 100 was 30% for WT GST–hGK Data
points and error bars represent the mean ± SD of three
indepen-dent experiments.
asl
asl
*
70
30 40 50 60
Model 1 Model 3
Time (ps)
10 20
Model 4
A
B
C
Fig 5 (A, B) Computed B-factor values and changes in the interdo-main cleft angle The computed B-factor values for the MD simu-lated model structures of the apoenzyme and the hGK–ATP binary complex The values are colour-coded onto the 3D ribbon structure
of (A) the apoenzyme and (B) the hGK–ATP binary complex, with red corresponding to the most mobile region (B-factor ‡ 400 A˚ 2
), blue corresponding to the most stable region (B-factor £ 40 A˚ 2 ), and green corresponding to B-factor values in the range 40–400 A˚2 Note also the change in secondary structure of the flexible active site loop (asl), comprising residues Ser151–Cys181, on binding of ATP (*) The B-factor values versus residue numbers are shown in Fig S2B (C) The changes in the interdomain cleft angle during the 2-ns MD simulations at 300 K The change in the cleft angle was defined by the residues Lys169–Gly223 (‘hinge’)–Gly229, compati-ble with a partial closure of 15 Model 1: hGK super-open con-formation (including coordinates for the Glu157–Asn179 loop) Model 2: hGK super-open conformation with inserted Glc Model 3: hGK super-open conformation with inserted ATP Model 4: hGK ter-nary complex with Glc and ATP.
Trang 7( 50) on MD simulations of Glc dissociation from
the binary hGK–Glc complex [29] A molecular
motion was further indicated by the dyndom
algo-rithm [30], with the coordinates obtained for the
ligand-free form and the hGK–ATP complex at the
end of the simulations (Figs 6B,C and S3; Table S2),
also indicating partial closure of the cleft ( 33) and
an apparent domain motion, which were less dramatic
than for the Glc-induced conformational transition
(Table S2) In the final structure of the binary complex
(Fig 6A; Table S1), the adenosine moiety is packed
between helices 12 and 15 in the L-domain [29] and
stabilized by hydrogen bonds (with Thr332 and Ser336
in helix 12) and hydrophobic interactions (with Val412
and Leu415 in helix 15)
A conformational change was also indicated by the
MD simulations of the modelled ternary hGK–Glc–
ATP complex In the final structure of the simulations,
the interactions of the adenosine moiety were similar
to those observed in the binary ATP complex, with the
a-phosphate and b-phosphate oxygen atoms forming
hydrogen bonds with Thr228 and Ser411 in the
L-domain (data not shown)
For comparison, when the MD simulations were
performed with Glc in the super-open conformation
(Fig 5C), no significant change in the interdomain
cleft was observed The substrate was found to be
positioned at the active site, as expected [18], including
the interactions with the primary contact residues
Asn204 and Asn231 (data not shown) However, no
interactions with Thr168 and Lys169 were seen, as the
Ser151–Val181 surface loop was not displaced in the
direction of Glc, and there was no measurable closure
of the active site cleft during the 2-ns MD simulations
(Fig 5C), as observed in the crystal structures of the
binary GK–Glc complex [18,31] Thus, in this case, the
simulation time (2 ns) was too short to demonstrate
the large global conformational transition observed by
ITF upon Glc binding, which has a millisecond to
minute time scale [18,25,32], characteristic of this
hys-teretic enzyme, and thus out of reach of
nanosecond-scale MD simulations
Steady-state kinetics
The steady-state kinetic properties of WT GST-hGK
were determined with Glc as the variable substrate at
high or low concentrations of MgATP (Table 3)
Posi-tive cooperativity was observed with respect to Glc at
5 mm (saturating) MgATP (nH= 1.95 ± 0.10)
(Fig 7A) with an [S]0.5 value of 8.2 ± 0.3 mm
How-ever, at 0.05 mm MgATP, the cooperativity was reduced
to nH= 1.07 ± 0.07 (Fig 7B), and the [S]0.5value was
A
**
B
C
D205
R447
K169
K296
T332
S336
L415 V412 S411
T228
ATP
[helix 12]
[helix 15]
αα
β γ
Fig 6 The ATP-binding site in the MD simulated model structure
of the binary hGK–ATP complex and the domain motion induced by ATP binding to the hGK apoenzyme (A) Close-up view of the ATP-binding site in the MD simulated model structure of the binary hGK-ATP complex, showing the main contact residues with ATP; for a presentation of all contact residues, see Table S1 For helix nomenclature, see [47] (B, C) The domain motion induced by ATP binding to the apoenzyme with partial closure of the active site cleft and a rotation angle of 33 The coordinates were those obtained for (B) the modelled super-open conformation, including the Glu157–Asn179 loop, and (C) the modelled open conformation with inserted ATP (GK–ATP) The Carmsd values were 4.01 A ˚ for the whole protein, 2.09 A ˚ for the fixed domain (349 residues), and 3.91 A ˚ for the moving domain (87 residues) The dynamic domains were identified with the DYNDOM program [30] The Cabackbone structures, shown in line presentation, were colour-coded as fol-lows: blue, fixed domain; red, moving domain; and green, connect-ing residues For comparison, the correspondconnect-ing data for the domain motion induced by Glc binding to the apoenzyme are shown in Table S2 **ATP.
Trang 8increased to 14.3 ± 1.7 mm The fact that the kinetic
cooperativity is dependent on the MgATP concentration
is consistent with previous data reported for the rat liver
isoform [33,34] With MgATP as the variable substrate,
a hyperbolic curve was obtained at a high Glc
concen-tration (60 mm), with a Kmof 0.16 ± 0.01 mm (Table 3;
Fig 7C) However, at a low Glc concentration
(0.5 mm), negative cooperativity was observed with
respect to MgATP (nH= 0.87 ± 0.06) (Fig 7D),
con-sistent with a previous report on the rat liver isoform
[34], and the Km was reduced to 0.04 ± 0.003 mm
Interestingly, the L146R mutant, with a severely
reduced affinity for Glc (Table 1), demonstrated similar
negative kinetic cooperativity with respect to MgATP as
the variable substrate (nH= 0.73 ± 0.04)
Discussion
The bisubstrate reaction catalysed by monomeric GK
is mechanistically characterized by diffusion-controlled
binding of Glc to thermodynamically favoured
ligand-free conformations of the enzyme (Scheme 1), followed
by global hysteretic isomerization of the enzyme to a
closed conformation [29,31]
From crystallographic, biophysical and kinetic
stud-ies on GK, it is known that both substrate binding
and catalysis require substantial conformational
changes in the enzyme Ligand-free hGK is
structur-ally dominated by a super-open conformation [31],
which, in the crystal structure, is locked in an inactive
state by electrostatic and hydrophobic interactions
between the C-terminal helix (helix 17) and helix 6
[18] Three residues (Asn204, Asn231, and Glu256) in
the large domain [31] function as primary contact
res-idues in the binding of Glc [18,31] Pre-steady-state
analyses of Glc binding to WT hGK [26,32,35] have
provided evidence that the ligand-free enzyme in
solu-tion is in a pre-existing equilibrium between at least
two conformers (marked as GK and GK„ in
Scheme 1), i.e the super-open conformation ( 80–
95%) and an alternative (presumably less open)
con-formation ( 5–20%) with a higher affinity for Glc [26,35], which adds to the kinetic complexity of this reaction Recent high-resolution NMR analyses and pre-steady-state Glc binding experiments also suggest that GK is capable of sampling multiple conforma-tional states, both in the absence and the presence of Glc [32,36] The global conformational changes trig-gered by Glc binding have been defined crystallo-graphically [31] In the closed conformation (marked
as GK* in Scheme 1), precise alignment of additional substrate contact residues (notably Thr168 and Lys169 in the flexible surface⁄ active site loop) [18,29] and the subsequent higher affinity for Glc efficiently accelerate the chemical reaction (k3) on binding of the cosubstrate MgATP The overall binding constant
K1 for Glc and the values for the forward (k2) and reverse (k)2) rates of the conformational transition, which probably includes intermediates [29,31,35,36], have been estimated by stopped-flow fluorescence spectroscopy [25] In that study, the GK– Glc M GK*–Glc interconversion was found to
be slow, with k2= 0.45 s)1 and k)2= 0.28 s)1 (K2= 1.6), favouring the forward rate and isomeriza-tion, whereas the isomerization was unfavourable with 2-deoxyglucose as the substrate (K2= 0.8) Here, we present experimental evidence that ATP binds to the ligand-free form, and that this also results in changes
in the protein conformation
ATP binds to the ligand-free open conformation
of hGK Previous attempts to demonstrate direct binding
of ATP to the ligand-free form of rat GK by ITF
Table 3 The kinetic constants for WT GST–hGK at high and low concentrations of the fixed substrate The catalytic activity was measured
at 37 C, as described in Experimental procedures Kinetic parameters were calculated by nonlinear regression analyses with the Hill and Michaelis–Menten equations.
Glc as variable substrate
ATP as variable substrate
GK‡ + Glc
GK‡ / ≠ ·Glc GK*·Glc GK*·Glc6P GK + Glc6P
GK≠ + Glc K1
≠
ADP MgATP
Scheme 1 Reaction scheme for mammalian glucokinase.
Trang 9spectroscopy [37] and hGK by differential scanning
calorimetry [25] were reported to be unsuccessful The
topic was more recently readdressed [26] with a
non-hydrolysable ATP analogue (AMP-PNP) and ITF,
and relatively large quenching of the fluorescence
sig-nal was demonstrated, interpreted as a
nucleotide-induced conformational change However, as no
cor-rections were made for a large inner filter effect,
owing to the significant absorbance of the nucleotide
at the excitation wavelength (285 nm), we have
cor-rected for this effect here (at kex= 295 nm) as well
as for any effect of nonspecific binding to the enzyme
(i.e not in the active site), with the non-ATP-binding
mutant form T228M (Table 1) as a reference enzyme
Our analyses revealed that AMP-PNP and ATP do
indeed bind to hGK (Fig 2) in the ligand-free open
conformation, and the MD simulations (Fig 6)
fur-ther support this conclusion and also show the
resi-dues (including the mutated residue Thr228) directly
contacting ATP at the active site of WT hGK
The partial quenching effect of AMP-PNP on the
T228M reference enzyme with disrupted ATP binding
at the active site (Fig 2A,C) suggests a contribution
of nonspecific binding of that nucleotide (i.e not in
the active site) in addition to its inner filter effect,
as observed for free Trp The idea that AMP, in contrast to MgADP)⁄ MgATP2), can bind to more than one site has been suggested for the rat liver isoform [16]
Binding of ATP to ligand-free hGK results in a conformational change
High-resolution NMR analyses [36] have revealed that
GK is an intrinsically mobile enzyme whose structure and dynamics are modulated by temperature and ligand binding Here, we provide the first experimental evidence of ATP-dependent structural changes in WT hGK Specifically, our ITF quenching (Fig 2) and
MD simulations (Figs 5 and 6) indicate a significant conformational change upon ATP binding, including motion of the flexible surface⁄ active site loop and par-tial closure of the active site cleft (Figs 5C, 6B,C and S3) A change in conformation is further supported
by the significant protective effect of ATP on binding
of the extrinsic fluorescence probe ANS (Fig 3A,B) and on the limited proteolysis by trypsin (Fig 3C) In both assay systems, Glc showed more potent inhibi-tion than ATP, which may be related to the larger conformational change and more effective closure of
A
C
B
D
Fig 7 Steady-state kinetic properties of WT GST–hGK with Glc and MgATP as the variable substrates (A) At 5 m M MgATP, positive coo-perativity with respect to Glc was observed (n H = 1.95 ± 0.10) (B) At a low (0.05 m M ) concentration of MgATP, the cooperativity with respect to Glc was reduced (nH= 1.07 ± 0.07) (C) At 60 m M Glc, the binding curve for MgATP was hyperbolic (nH= 1.15 ± 0.04) (D) At a low (0.5 m M ) concentration of Glc, negative cooperativity with respect to MgATP binding was observed (nH= 0.87 ± 0.06) For all nonlinear regressions, the correlation coefficient (r 2 ) was > 0.99 The steady-state kinetic constants are summarized in Table 3.
Trang 10the active site cleft induced by Glc binding (Fig S3;
Table S2)
Kinetic cooperativity with respect to Glc
In general, the mechanism for cooperativity observed
in enzyme kinetic studies represents an experimental
challenge For monomeric GK, several models have
been considered to explain the positive kinetic
cooper-ativity with respect to Glc, including: (a) a random
order mechanism of substrate (Glc and MgATP2))
addition [13,38]; and (b) a sequential order mechanism
[15,39,40], in which the binding of Glc as the first
sub-strate induces a slow, concentration-dependent
confor-mational transition [34,40] characteristic of a hysteretic
enzyme [41,42] (Scheme 1) The Glc-induced
multipha-sic ITF enhancement (millisecond to minute time scale)
of WT and mutant forms of GK [18,25,26,35,37,43]
strongly favours the second mechanism, and support
the idea that the cooperativity can be explained by an
equilibrium between conformational states with
differ-ent affinities for Glc [18,25,26,35,37,43] However, little
experimental effort has been made to include or
exclude any contribution of (Mg)ATP binding to the
kinetic cooperativity
The ligand-free and the binary GK–Glc complex are
dynamic entities [32,36], and binding of (Mg)ATP may
shift the equilibrium between different enzyme
confor-mations, as shown for Glc [18,25,26,35,37,43] In this
study, the binding of ATP to the ligand-free enzyme
was found, by four independent criteria, to trigger
con-formational changes, including partial closure of the
active site cleft (Figs 5B, 6B,C and S3) Moreover,
pre-vious [34] and present (Table 1) steady-state kinetic
analyses are also compatible with conformational
con-trol of GK catalytic activity by the binding of
(Mg)ATP, with possible implications for kinetic
coo-perativity with respect to Glc Thus, the coocoo-perativity
is largely reduced (nH= 1.07 ± 0.07) at low
concen-trations of MgATP (Fig 7B; Table 1) [34] and when
MgATP is replaced by MgITP, a poor phosphoryl
donor ATP analogue [44]
In most previous steady-state kinetic analyses, rat GK
(liver) was observed to be noncooperative with respect
to (Mg)ATP [33,45,46] However, Neet et al [34]
reported negative kinetic cooperativity (nH= 0.84)
when measurements were made in the presence of 30%
glycerol at a low Glc concentration (0.5 mm), and this
was also observed here for the recombinant pancreatic
hGK in the absence of glycerol (nH= 0.87 ± 0.06)
However, when hGK activity was measured at high
glu-cose concentrations, the Hill coefficient for (Mg)ATP
approached 1.0 (Fig 7C), as expected from studies on
rat liver GK [33,45,46] Negative cooperativity (nH= 0.73 ± 0.04) was also observed for the GCK-MODY mutant L146R, which has severely reduced affinity for Glc (Table 1) Moreover, our studies on this mutant revealed that the analogue ATPcS (at 2.5 mm) increases the mutant’s low equilibrium binding affinity for Glc (Kd decreases from 19.3 ± 3.8 mm to 14.0 ± 1.4 mm), as well as the Glc-induced fluorescence enhancement (by 25%) (Fig 4) These effects may be related to partial catalytic activation following (Mg)ATP binding at physiological concentrations of Glc Similar or possibly larger effects of ATP in promot-ing a catalytically competent state may occur in other mutations associated with GCK-MODY
Conclusions
Using biochemical and biophysical methods, we have obtained experimental evidence in support of binding
of ATP to the ligand-free hGK, resulting in a change
of protein conformation The MD simulations indicate that the binding triggers molecular motion of the flexi-ble surface⁄ active site loop and partial closure of the interdomain active site cleft The modelled structure of the hGK–ATP binary complex shows the residue con-tacts involved in ATP binding at the active site Our findings further support conformational regulation of
GK by ATP binding, with possible implications for kinetic cooperativity with respect to Glc Further mutational studies, notably of GCK-MODY-associated mutations, may contribute to a better understanding
of the mechanistic and functional implications of the multiple conformational equilibria and the conforma-tional transitions induced by both Glc and (Mg)ATP, with possible future clinical implications
Experimental procedures
Materials
The oligonucleotide primers used for site-directed mutagen-esis were from Invitrogen (Carlsbad, CA, USA) The QuickChange XL Site-directed Mutagenesis Kit was from Stratagene (La Jolla, CA, USA) Glutathione Sepharose 4B was from Amersham Biosciences (GE Healthcare Europe GMBH, Oslo, Norway) Glc was from Calbiochem (San Diego, CA, USA) Magnesium chloride, magnesium ace-tate, guanidine hydrochloride, trypsin (bovine pancreas), trypsin inhibitor (soybean), pyruvate kinase (rabbit muscle), phospho(enol)pyruvate, ATP, ANS and AMP-PNP were from Sigma-Aldrich (St Louis, MO, USA) ATPcS was obtained from Roche Diagnostics Corporation (Indianapo-lis, IN, USA) All chemicals and buffers used for fluores-cence measurements were of the highest analytical grade